Droplet-based technologies were developed in recent years by applying existing technologies. Functional units include production (WO2002/068104 A1, WO2005/089921) and storage (WO2010/042744 A1), droplet splitting and fusion (US2012/0108721 A1), injection of additional reagents (WO2010/151776 A2), and deflection/sorting (U.S. Pat. No. 8,765,455 B2, US 20130213488 A1).
As an example, one may attempt to select particular individual microorganisms out of a population of millions of microorganisms with respect to a specific activity (e.g. growth rate or biosynthesis of a product). In the classic approach, the cell population is distributed in a suitable solution over a solid nutrient medium and isolated in this way. The individual colonies that have grown are examined after a variable incubation phase which can typically last from days to weeks. This step of cultivating and harvesting large numbers of individual colonies is very time-consuming and cost-intensive. Advanced robot systems can bring significant time savings to this task, but the amount of working hours needed is still immense. Droplet-based microfluidics, however, is a suitable tool for solving this issue. In this case the microorganisms are spatially separated from one another in aqueous droplets of variable size. In combination with a surfactant and an oil phase located outside the droplet, such emulsion droplets, typically in the picoliter range can be handled easily. They can be analyzed in a continuous microfluidic flow using currently available technology. The droplets with the microorganisms can be sorted at rates in the kilohertz range (Baret et al., Lab-on-a-Chip 2009, 9, 1850-1858).
The resulting effects to use aqueous droplets after sorting have however been inefficient in the prior art, because individual extraction is difficult to handle. In the prior art the microfluidic droplets are collected and the aqueous phases of all droplets are combined using emulsion dissolving agents. Thereby the content of the droplets is separated from the oil phase and the enclosed cells are recultivated as a whole (Mazutis et al., Nature Protocols, 2013, pp. 870-891, Najah et al., Chemistry & Biology 2014, Vol 21/12, pp. 1722-1732, Debs et al., PNAS, 2012, Vol. 109, pp. 11570-11575, and others). A retrieval of a single unlabeled aqueous emulsion droplet in the picoliter volume range is not possible with methods or devices of the prior art.
Bai et al. in 2014 describe a high throughput sorting and depositing of agarose droplets by a FACS (fluorescence activated cell sorter), (Y. Bai et al., Sensors and Actuators B: Chemical, 2014, 194, pp. 249-254). This approach has however several disadvantages. It requires the use of gel-like droplets and the purchase of very expensive FACS devices. Moreover, sorting in the case of FACS necessitates the use of fluorescent markers expressed and localized at sufficient levels within the single cells, further limiting the biological applications of these devices.
A system has been published that detects aqueous droplets in a continuous oil phase within a capillary and deposits them on a MALDI (matrix assisted laser desorption ionization) plate (Küster et al., Analytical Chemistry, 2013, 85, pp. 1285-1289). However, in this case only large droplets of a minimal size of 3 nl can be isolated and the method is restricted to the deposition of periodically generated droplets. Moreover the subsequent analysis of the droplets is restricted to mass spectroscopy in which the biological content is typically destroyed.
In the prior art it has therefore not been possible to isolate individual microfluidic emulsion droplets on the picoliter scale from a droplet pool in a microfluidic system especially when droplets are produced for an isolation at varying irregular frequencies. Moreover a subsequent cultivation of target organisms that have been encapsulated in a microfluidic emulsion droplet outside the microfluidic system has also not been possible.